Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.
Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein ( EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.[1]References
- Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish. Hsiao, C.D., Hsieh, F.J., Tsai, H.J. Dev. Dyn. (2001) [Pubmed]
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